Unit 7 Flashcards
MOLECULAR DIAGNOSIS OF INFECTIOUS DISEASES
Infectious diseases involve:
○ Viruses
○ Bacteria
○ Fungi
○ detect parasites
- Molecular characterization of microorganisms.
- Development and evaluation of molecular-based laboratory tests of clinical specimens isolated in cultures.
- Comparison of biochemically similar organisms in outbreak situations (known as molecular epidemiology) to ascertain whether the isolates have a common independent source.
APPLICATIONS OF MOLECULAR TECHNOLOGY
APPLICATIONS OF MOLECULAR TECHNOLOGY
- molecular characterization
- for molecular based laboratory tests
- molecular epidemiology
Comparison of biochemically similar organisms in outbreak situations is known as?
molecular epidemiology
Example of molecular epidemiology
SARSCOV (alpha, beta, delta, and omicron strains), slightly different genomes
Analyte for molecular testing used in PCR, RT-PCR, LAMP etc.
genome
mRNA transcript (basis for gene expression)
Transcriptome
proteins of an organism (this is not targeted by molecular tests as much as genome)
Proteum
Proteum is used in which detection method?
MALDI-TOF (Laser desorption targets protein»ionizes»charged)
Pure culture/colony is used in MALDI-TOF
TRUE (ie proteum)
- Time-consuming to isolate organisms
- Hazardous with which to work in the clinical laboratory
- Reliable laboratory tests were lacking
- Organisms that are received in clinical laboratories in high volumes
- Genes that confer resistance to antimicrobial agents
- Characterization of DNA, RNA, and protein to find and identify new organisms and to further characterize or classify known organisms.
COMMON SPECIFIC TARGETS
Hazardous with which to work in the clinical
laboratory
systemic fungi (histoplasma/coccidioides); dangerous to grow in the lab kaya mas safer if molecularly tested
Time-consuming to isolate organisms
TB = takes 8 weeks to be isolated
Reliable laboratory tests were lacking
Hepatitis C
■ Culturing viruses in laboratories is
impractical and costly
■ Can be detected through genomic
testing, protein testing
(antigen/antibody testing)
Organisms that are received in clinical laboratories in high volumes
Streptococcus pyogenes, Chlamydia trachomatis, neisseria gonorrhoeae (last
two often undergo PCR in the Philippines)
ginagamit for transport and collection of swab sample dahil mas maliit yung swab nya; less painful
Stuart transport medium
Genes that confer resistance to antimicrobial agents
Methicillin-, Oxacillin-, Vancomycin-resistant
VRSA Genes
Van A, Van B, Van C, enterococcus
resistance to vancomycin
MRSA Gene
mecA and mecC
■ Staphylococcus
■ Klebsiella
■ Acinetobacter
■ Pseudomonas
■ Enterococcus
1. E. faecalis
2. E. faecium
S.K.A.P.E = To watch out organisms in
cultures; “super bombs”; drug resistant
resistance to rifampicin gene
rpoB (RNA polymerase B gene)
- OR specimens = kapag nilalagyan ng formalin, kasi di mo maeextract yung DNA
- Lysed na yung cells
- Highly fragmented na yung nucleic acids
Rejected samples
Characterization of DNA, RNA, and protein to find and identify new organisms and to further
characterize or classify known organisms.
influenza virus and Sars-CoV
- Viability is not as critical for most molecular testing
- Quality of nucleic acids may be compromised if the specimen is improperly handled
- DNA and especially RNA will be damaged in lysed or nonviable cells
- Avoid contamination that could yield false-positive results
SPECIMEN COLLECTION
only formalinized tissue we use for DNA or RNA extraction
Formalin fixed paraffin embedded tissue
pseudoperoxidase activity in sputum with blood affects downstream application of PCR
TRUE
DNA and especially RNA will be damaged in lysed or nonviable cells because of possible release of nucleases
TRUE
component of sputum with blood that inhibits taq polymerase in PCR
Hemoglobin
PCR IS VERY POWERFUL, but it can saturate the process if very small amount of DNA is used
TRUE
LLOD - Lower limit of detection
amount of DNA used; low LLOD is good! mas sensitive
Principle: Real-time monitoring of PCR progression was performed using fluorescence signal accumulation
Quantitative PCR
Principle: Isothermal Amplification was performed using four specific primers in the presence of Bacillus
Loop-mediated Isothermal Amplification
Principle: Amplification reactions were performed for individual nucleic acid molecules in a separate space
Digital PCR
Principle: The different melting temperatures of mononucleotides lead to different melting curves
High-resolution Melting
Applicability: Routine quantitative detrection of common pathogens such as novel coronavirus, HBV, human papillomavirus, and others in the laboratory
Quantitative PCR
Applicability: Absolute quantification of low-content pathogens such as HBV, HIV, and MTB, etc.
Digital PCR
Applicability: Genotyping of pathogens such as Escherichia coli, Staphylococcus aureus, adenoviruses, etc.
High-resolution Melting
Advantage: Absolute quantification, low sample requirement, and high tolerability
Digital PCR
Applicability: Primary field screening of pathogens such as MTB and Plasmodium.
Loop-mediated Isothermal Amplification
Advantage: Good specificity, high sensitivity, high degree of automation, and low cost.
Quantitative PCR
Advantage: Rapid, high throughput, short time, and low cost
High-resolution Melting
Limitations: Numerous interference factors, time-consuming, instruments are expensive
Quantitative PCR
Limitations: High cost, limited throughput, complex operation
Digital PCR
Advantage: Low instrument requirements, fast reaction speed, low cost, and high sample
Loop-mediated Isothermal Amplification
Limitations: Complex primer design, non-specific amplification, low throughput
Loop-mediated Isothermal Amplification
Limitations: High requirement for uniformity of temperature and primer design
High-resolution Melting
False-positives: Nucleic acid contamination, non-specific amplification
False-negatives: Inhibitors, enzyme inactivation, too little template
Quantitative PCR
False-positive: Nucleic acid contamination
Digital PCR
False-positive: Non-specific amplification
High-resolution Melting
False-positive: Cross-contamination
Loop-mediated Isothermal Amplification
Best dye?
HRM
HRM > Evagreen > SyBr Green 1
TRUE
- Negative derivative in fluorescence and temperature, dalawang peaks ang nakita, edi HRM daw siya
- Means 2 targets yung naamplify mo
- If white peak = naamplify and may nonspecific amplification
TRUE
Polycythemia vera gene
JAK2 (V671E to V617F)
PCR (RT,qRT etc)
DNA Sequencing
Pulse Gel
Electrophoresis
MALDI TOF (mass spectrometry principle)
Molecular methods
- PCR-based amplification.
- can overcome low sensitivity to improve the specificity of conventional phenotypic tests.
- provide early diagnosis for proper treatment with potential reduction in mortality and morbidity.
- all PCR assays have been designed to detect a low load of a given microorganism among a huge number of human cells.
NUCLEIC ACID AMPLIFICATION TECHNIQUES
for MTB/RIF, MRSA, HBV, HIV, HCV, CT/NG (Chlamydia and Neisseria), HPV, SARS-CoV-2, C. difficile, etc.
Cepheid GeneXpert® Systems
Most significant sexually transmitted HPV is 16 and 18
TRUE
<100 CFU/mL = cannot be detected
TRUE
for respiratory, gastrointestinal and cerebrospinal organisms
BIOFIRE® FILMARRAY® SYSTEMS
for HIV, HBV and HCV
Roche COBAS® TaqMan® viral loading systems
Detects through electrophoresis
Seegene AnyplexII HPV HR Detection
- Conserved regions are ideal primer targets for amplification from all bacterial species.
- Measuring the degree of divergence observed within the 16S rRNA molecule.
- It contains well contained regions and variable regions
- The signature sequence obtained is compared to a database containing sequences of known microorganisms.
- The number of similar nucleotide bases between sequences is used to calculate the percent identity and ascertain the identification of the microorganism.
DIAGNOSIS WITH SEQUENCING
- The rRNA genes and their intergenic regions found in bacteria are commonly used for prokaryotic phylogenetic studies.
- The small-subunit rRNA molecule is a fragment with a sedimentation coefficient of 16S and is encoded by a roughly 1542-bp gene.
- 16S is also used for community analysis = gold standard
TRUE FOR DIAGNOSIS WITH SEQUENCING
(beta subunit of RNA polymerase)
rpoB
(manganese-dependent superoxide dismutase)
sodA
(elongation factor Tu)
tuf
- rpoB (beta subunit of RNA polymerase)
- sodA (manganese-dependent superoxide dismutase)
- gyrA or gyrB
- tuf (elongation factor Tu)
- recA
- secA
- Heat shock proteins.
Other DNA targets have been used to better separate closed related species include:
Similar to 16S rRNA gene, these alternative DNA targets have conserved regions flanking variable regions that can be used to differentiate closely related bacterial species.
TRUE